Abstract: The assembly of functional biological materials requires precise control over the synthesis and deposition of their constituent polymers. In plants, specialized cells gain exceptional strength from their secondary cell walls (SCWs), though the mechanisms ensuring the coordinated production of SCW components remain poorly understood. Using cotton fiber as a model for massive cellulose deposition, we uncover a multi-tiered transcriptional cascade involving the R2R3-MYB transcription factor GhMYB44. We show that GhMYB44 functions as a positive regulator that directly activates the pivotal NAC factor, GhFSN43, which in turn commands key cellulose synthesis genes, establishing a complete regulatory module from an upstream MYB to downstream structural genes. Crucially, we reveal that this entire pathway is potentiated at two distinct nodes through protein–protein interactions: an upstream GhMYB44–GhREV5 complex enhances the initial signal, while a downstream GhFSN43–GhFSN1 complex functions cooperatively to strengthen downstream transcriptional activation. Our findings suggest that GhMYB44 participates in a multi-tiered regulatory module where transcriptional output is enhanced by layered synergistic protein–protein interactions. This work clarifies a key regulatory pathway controlling SCW biosynthesis and offers novel targets for improving cotton fiber quality.

Key words: cellulose biosynthesis, cotton ?ber, GhFSN43,GhMYB44, secondary cell wall (SCW), transcription factor (TF)